Dimerization and N-terminal domain proximity underlie the function of the molecular chaperone heat shock protein 90

Abstract

Heat shock protein (hsp)90 functions in a complex chaperoning pathway where its activity is modulated by ATP and by interaction with several co-chaperones. One co-chaperone, p23, binds selectively to the ATP-bound state of hsp90. However, the isolated ATP-binding domain of hsp90 does not bind p23. In an effort to identify the p23-binding domain, we have constructed a series of hsp90 deletion mutants fused with glutathione-S-transferase (GST). Full-length GST-hsp90 is able to bind p23, and also, to chaperone assembly of progesterone receptor complexes. Truncations from the C terminus of GST-hsp90 reveal a C-terminal boundary for the p23-binding domain at approximately residue 490. This fragment contains, in order, the ATP-binding domain, a highly charged region, and 203 residues beyond the charged region. p23 binding is unaffected by deletion of the charged region, indicating that two noncontiguous regions of hsp90 are involved in p23 binding. These regions are only effective when hsp90 is in a dimeric state as shown by loss of p23 binding upon removal of GST or as shown by use of FK506-binding protein12-hsp90 constructs that form dimers and bind p23 only in the presence of a bivalent drug. Thus, p23 binding requires an hsp90 dimer with close proximity between N-terminal regions of hsp90 and a conformation specified by ATP.H eat shock protein (hsp)90 is an abundant and ubiquitous molecular chaperone that is required to assist the conformational maturation of specific targets involved in many key functions of the cell such as cell-cycle regulation and signal transduction. Among hsp90 targets are several protein kinases such as v-Src, Weel, and c-Raf, transcriptional regulators such as p53 and steroid receptors, and two polymerases: that of hepatitis B virus and telomerase (for review, see refs. 1-4). To date, the best known maturation process driven by hsp90 is the assembly of steroid receptors into a high-affinity hormone-binding conformation. This complex, multistep process occurs in an ATPdependent manner and involves several other chaperones and co-chaperones (4). Three dynamic steps have been observed in this assembly process (5-7). The receptor initially associates with hsp70, assisted by hsp40. This association develops into an intermediate complex in which the co-chaperone, hsp organizing protein (Hop), associates with both hsp70 on the receptor and hsp90, while the protein Hip binds to hsp70. As the receptor complex matures, hsp90 bound to the receptor interacts with p23 and any one of four tetratricopeptide repeat-containing proteins, FK506-binding protein (FKBP)52, FKBP51, cyclophilin-40, or phosphoprotein phosphatase 5. The details of the interactions between these partner proteins are poorly understood and the mechanisms driving the transitions from one complex to another are still unresolved.hsp90 is a dimeric protein with multiple domains. Several proteins involved in the hsp90 chaperone pathway possess structurally related tetratricopeptide repeat motifs that mediated their interaction with hsp90 a...

Molecular Imaging of the Efficacy of Heat Shock Protein 90 Inhibitors in Living Subjects

“…The specificity of using NRL(M185V)-p23/ Hsp90h-CRL was confirmed using Hsp90h2.2(K107A)-CRL, Hsp90h2.2(K111A)-CRL, and Hsp90h2.2(K107AK111A)-CRL mutant fusion constructs. These mutations are analogous to the K111A mutation in chicken Hsp90a that reduces binding to geldanamycin (29). Figure 3A (Fig.…”

Ligand-induced Conformational Shift in the N-terminal Domain of GRP94, an Hsp90 Chaperone

“…Experimentally, N-domain proximity and nucleotide binding correlate with p23 binding to Hsp90 (44). In addition, fluorescence emission spectra of pyrene-derivatized Hsp90 (21) and electron microscopy of antibody decorated Hsp90 (45) also suggested N-domain proximity in the presence of ATP.…”

Coordinated ATP Hydrolysis by the Hsp90 Dimer

“…The functional consequences of these changes are still largely unclear. One of the few functional correlations of these rearrangements is that binding of the cofactor p23 requires ATP bound to Hsp90 (28) and the dimeric form of the N-terminal domains (22,29).…”

“…Here, cross-linking data indicate that the N-terminal domains associate in the presence of AMP-PNP, which had been suggested previously based on electron microscopic data (26). This seems to be a prerequisite for the association of Hsp90 with the co-chaperone p23, which is known to occur after ATP-binding (27,28,29). In the studies of Weikl et al (22) and Prodromou et al (25), fragments of Hsp90 lacking C-terminal domains were found to be considerably less active than wild-type Hsp90.…”